Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of Pichia pastoris

Results We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation. Conclusion Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR

Rapid screening of cellular stress responses in recombinant Pichia pastoris strains using metabolite profiling

Heterologous protein production in the yeast Pichia pastoris can be limited by biological responses to high expression levels; the unfolded protein response (UPR) is a key determinant of the success of protein production in this organism. Here, we used untargeted NMR metabolic profiling (metabolomics) of a number of different recombinant strains, carried out in a miniaturized format suitable for screening-level experiments. We identified a number of metabolites (from both cell extracts and supernatants) which correlated well with UPR-relevant gene transcripts, and so could be potential biomarkers for future high-throughput screening of large numbers of P. pastoris clones.This study was wholly funded by the Bioprocessing Research Industry Club (BRIC), a partnership between BBSRC, EPSRC and a consortium of leading companies (http://www. bbsrc.ac.uk/business/collaborative-research/industry-clubs/bric/background.aspx, Grant reference BBF0049071)

Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of <i>Pichia pastoris</i>

<div><p>Results</p><p>We have followed a typical fed-batch induction regime for heterologous protein production under the control of the <i>AOX1</i> promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the <i>TRY1</i> gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation.</p><p>Conclusion</p><p>Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR.</p></div

The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data

Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris

Expression of <i>HAC1</i> during fed-batch culture, 0 (before), 2 and 4 hours after the start of methanol addition to wild-type GS115 (black), <i>TRY1-1</i>(blue) and <i>TRY1-3</i> (red), strains containing 1 and 3 gene copies of the human typsinogen gene, respectively, under the control of the <i>AOX1</i> promoter.

<p>Error bars show SEM.</p

Expression of the gene encoding D-arabitol-2-dehydrogenase (top) and “<i>YDL124W”</i> (bottom), 0 (before), 2 and 4 hours after the start of methanol addition to wild-type GS115 (black), <i>TRY1-1</i>(blue) and <i>TRY1-3</i> (red), strains containing 1 and 3 gene copies of the human typsinogen gene, respectively, under the control of the <i>AOX1</i> promoter.

<p>Error bars show SEM.</p

Metabolite accumulation profiles during fed-batch culture from 0 (before) to 24 hours after the start of methanol addition to wild-type GS115 (black), <i>TRY1-1</i>(blue) and <i>TRY1-3</i> (red), strains containing 1 and 3 gene copies of the human typsinogen gene, respectively, under the control of the <i>AOX1</i> promoter.

<p>From top to bottom, panels represent a) formate (external), b) arabitol (external), c) trehalose (external), d) α / β-D-glucose (external), e) lactate (internal). Error bars show SEM.</p

The number of unique probes on the micro-array per gene.

<p>A total of 3371 genes were targeted by 9 unique probes, with the average length of these genes being 1844 bases.</p><p>The number of unique probes on the micro-array per gene.</p

Expression of <i>PCK1</i>, <i>PYC</i> and <i>PYK</i> during fed-batch culture, 0 (before), 2 and 4 hours after the start of methanol addition to wild-type GS115 (black), <i>TRY1</i>-1 (blue) and <i>TRY1</i>-3 (red).

<p>Error bars show SEM.</p